This study evaluated the adhesion and biofilm formation of Alicyclobacillus acidoterrestris on industrial orange juice processing equipment and the bactericidal efficacy of peracetic acid, sodium hypochlorite, and quaternary ammonia after biofilm formation. The efficacy of these sanitizers against the spores of this microorganism was also evaluated. Stainless steel and nylon surfaces exhibited higher cell adhesion levels than did polyvinyl chloride surfaces. Peracetic acid was the most effective in removing biofilms from all surfaces (P < 0.05) and also reduced bacterial counts by 3 log CFU/cm² on the surface of polyvinyl chloride, but the other sanitizers also reduced the bacterial counts by 2 log CFU/cm². Quaternary ammonia exhibited the optimal minimum sporicidal concentration, preventing spore germination after only 15 s of contact at a concentration of 82 ppm. The flow cytometry results indicated that the spores and cells had low incidences of plasma membrane lysis after treatment with sanitizer, suggesting that lysis is not the principal mode of action for these sanitizers on A. acidoterrestris.